PROJECT SUMMARY Cirrhosis-associated death is one of the leading causes of death in the United States and alcoholic fatty liver disease (AFLD) accounts for 48% of these deaths1. Despite its profound health and economic impact, the management of AFLD remains a challenging prospect because: (i) there are no effective diagnostic tools or biomarkers to assess individual susceptibility to AFLD development or progression to more severe clinical conditions, and (ii) no new therapeutic entities have been developed in the past four decades. The long term goal of this research project is to elucidate the redox-associated mechanisms involved in protecting the liver against AFLD and thereby identify potential novel preventive and/or therapeutic targets against this disease. Oxidative stress plays a central role in many pathways involved in the pathogenesis of AFLD. A major factor contributing to the development of oxidative stress is the depletion of glutathione (GSH), the most abundant non-protein thiol in the liver. Our preliminary studies using GCLM knockout (KO) mice demonstrate that ~85% deficiency in hepatic GSH renders mice protected from steatosis induced by chronic ethanol administration. These mice also exhibit: (i) enhanced capacity to metabolize ethanol and acetaldehyde, (ii) persistent oxidative stress and induction of nuclear factor-erythroid 2?related factor 2 (NRF2) target genes, and (iii) importantly, sustained activation of the AMP- activated protein kinase (AMPK? pathway and associated changes in lipid metabolizing genes. This research project will utilize the GCLM KO model to expand upon our preliminary studies and investigate our working hypothesis that chronic GSH depletion induces redox activation of the AMPK pathway that serves as the central link triggering protective mechanisms that prevent AFLD. We propose to: elucidate redox-associated mechanism(s) sustaining AMPK activation in KO hepatocytes, identify AMPK catalytic subunit isoform- dependent pathways involved in ethanol-associated metabolic and stress response in KO hepatocytes, and determine the contribution of hepatic versus extrahepatic effects of GSH deficiency in modulating AMPK pathway and the protective phenotype of KO mice. The findings from these studies will provide important mechanistic information regarding key signaling and metabolic pathways involved in protection against alcohol-induced liver damage. It is anticipated that such new knowledge will reveal novel therapeutic targets for the treatment of alcohol-induced liver injury, such as AFLD. This K01 Award will allow the applicant to acquire advanced knowledge and research competency in alcohol research through an integration of interdisciplinary resources. The applicant has assembled an advisory committee composed of an outstanding group of mentors and consultants. Dr. Vasilis Vasiliou (primary mentor) is a recognized leader in the field of ethanol metabolism and toxicity. Dr. Wajahat Zafar Mehal (co-mentor) is a renowned hepatologist and liver immunologist who has extensive expertise in alcoholic and non-alcoholic fatty liver disease. Dr. Michael Harris Nathanson (co- mentor) is the Director of the Yale Liver Center and is among the world leaders in studying signaling pathways in liver disease. The program will enlist the expertise of Dr. Hongyu Zhao (consultant), who is among the world leaders in the application of statistical methods in molecular biology. Each member of the advisory committee has a formidable record of training academically successful independent scientists and they will mentor the applicant's career and academic development as well as the implementation of proposed experiments.